RNAlater-compatible protocol for nuclei isolation from radical prostatectomy prostate cancer resections, enabling single-nucleus resolution RNA-seq.

We present an optimised protocol for single-nucleus isolation from RNAlater-preserved prostate cancer (PC) resections that enables transcriptomically robust snRNA-seq. Our streamlined procedure employs bead-mill homogenization and high-salt lysis to generate nuclei suspensions, eliminating the need for FACS or density-gradient purification. The protocol yielded high-quality libraries (Q30 ≥ 90%) with minimal ambient RNA. Analysis of 3,365 nuclei identified eight distinct cellular compartments, preserving the full cellular repertoire, including luminal, stromal, and immune lineages. These findings demonstrate that snRNA-seq is feasible in RNAlater-preserved PC specimens enabling routine analysis of clinical samples and expanding the applicability of single-cell genomics in translational research.